Antibiotic agents from S. coeruleorubidus, rubidus

ABSTRACT

This invention relates to new polypeptide antibiotics designated LL-BO2964 alpha , LL-BO2964 beta , LL-BO2964 gamma , and a mixture thereof designated LL-BO2964, produced during microbiological fermentation, under controlled conditions, using a new strain of Streptomyces coeruleorubidus subspecies rubidus or a mutant thereof. The new antibiotics are active against a variety of microorganisms and are also useful in treating tuberculosis.

BACKGROUND OF THE INVENTION

This invention relates to new antibiotics designated LL-BO2964α,LL-BO2964β, LL-BO2964γ and LL-BO2964, and to their production byfermentation.

This invention also relates to a new strain of Streptomycescoeruleorubidus subspecies rubidus and mutants thereof which producepolypeptide antibiotics.

The new antibiotics of the invention appear to be similar to the familyof polypeptide compounds such as antibiotic 6798RP disclosed in BritishPat. No. 846,801; antibiotic FR3383 disclosed in unexamined Japanesepatent applications No. 1054-988 (Derwent Abstract No. 49101X) and No.2093-701 (Derwent Abstract No. 66033Y) and Japan Kokai No. 77-93,701;and the A-38533 antibiotics disclosed in U.S. Pat. No. 4,180,564 and in"A-38533, A New Antipseudomonal Antibiotic: Fermentation, Isolation, andStructure Studies", Current Chemotherapy and Infectious Disease, Abs.No. 1032, 19th Interscience Conf., October 1979. The antibiotics of thepresent invention, however, show distinguishing physical characteristicsover these polypeptide compounds, including but not limited todifferences in elemental analysis, UV spectrum, and high pressure liquidchromatographic retention times.

SUMMARY OF THE INVENTION

The new antibiotics of the invention are arbitrarily designatedLL-BO2964α, LL-BO2964β, and LL-BO2964γ. The designation LL-BO2964indicates a mixture in any proportion of two or more of the α, β, and γcomponents and this designation with the identities of the includedcomponents will be used herein whenever a mixture of the components isdiscussed.

The antibiotics are present as an associated mixture when produced by amicrobial culture fermentation. The ratios of components in theassociated mixture will vary depending on the fermentation conditionsand the culture strain used. Typically, the antibiotics are formed bycultivation of a new strain of Streptomyces coeruleorubidus subspeciesrubidus or by cultivation of a mutant thereof, having accession numbersNRRL 12372 and NRRL 12373 respectively.

The antibiotics LL-BO2964α, β, and γ are solid, optically active,amphoteric polypeptides having the characteristic, significant carbon-13nuclear magnetic resonance absorptions presented in Table I, infra.These absorptions uniquely characterize each antibiotic anddifferentiate them from other polypeptide antibiotics. The antibioticsof the invention also exhibit the ultraviolet, visible, infrared, protonand carbon-13 nuclear magnetic resonance spectra depicted in FIGS. 1through 15. The elemental analyses for the α, β, and γ antibiotics ofthe invention show the presence of carbon, hydrogen and nitrogen but nosulfur. Based upon hydrolysis data, the antibiotics of the invention atleast contain one residue each of uracil, glycine, alanine,beta-alanine, 2-amino-3-N-methylaminobutyric acid, and m-tyrosine.

The pharmacologically acceptable acid addition salts of the amphotericpolypeptide antibiotics are also included within the invention. Theacids forming such salts are those pharmacologically acceptable organicand inorganic acid reagents which will at least in part complex thepolypeptide antibiotics of the invention but will not destroy them.

The antibiotics of the invention show in vitro and in vivo antibacterialactivity as well as antitubercular activity.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings depict the ultraviolet (UV), visible (Vis), infrared (IR)and nuclear magnetic resonance (proton or ¹ HNMR, and carbon-13 or ¹³CNMR) spectra of the new antibiotics LL-BO2964α, LL-BO2964β, andLL-BO2964γ in their substantially pure forms. Chart A correlates FIGS. 1through 15 with these individual antibiotics.

                  CHART A                                                         ______________________________________                                        UV-VIS, IR, .sup.1 HNMR AND .sup.13 CNMR Spectra of                           Individual Antibiotics                                                                 UV-VIS            .sup.1 HNMR                                                                            .sup.13 CNMR                              Antibiotic                                                                             (water)  IR(KBr)  (DMSO-d.sub.6)                                                                         (DMSO-d.sub.6)                            ______________________________________                                        LL-BO2964α                                                                       FIG. 2   FIG. 1   FIGS. 3, 4                                                                             FIG. 5                                    LL-BO2964β                                                                        FIG. 7   FIG. 6   FIGS. 8, 9                                                                             FIG. 10                                   LL-BO2964γ                                                                       FIG. 12  FIG. 11  FIGS. 13, 14                                                                           FIG. 15                                   ______________________________________                                    

DETAILED DESCRIPTION OF THE INVENTION

The new antibiotics LL-BO2964α, LL-BO2964β, and LL-BO2964γ are closelyrelated polypeptides produced by fermentation of a new strain ofStreptomyces coeruleorubidus subspecies rubidus and a mutant thereof.They are recovered from the fermentation broth as a mixture and theratio of the individual components in the mixture varies depending onthe fermentation conditions. In general, LL-BO9264γ will be the majorcomponent found in the antibiotic mixture typically produced. Isolationof the component mixture LL-BO2964 from fermented culture followed bychromatographic or other known method of separation produces grosslyseparated component fractions. The fractions can be further purified byknown methods to produce substantially pure primary componentsLL-BO2964α, β, and γ. Although the grossly separated component fractionseach overwhelmingly constitute one primary component, the γ componentfraction in this somewhat impure state appears to contain two additionalsubcomponents. These subcomponents are believed to be bound in complexform with the primary component, LL-BO2964γ, and are also polypeptideantibiotics. They are present in minor amounts and have a compositionand structure which appears to be very close to that of the primarycomponent LL-BO2964γ. The α and β component fraction, in this somewhatimpure state also appear to be complexed with minor amounts ofstructurally similar polypeptide subcomponents. In general, however, theprimary components, LL-BO2964α, β, and γ will be present in anoverwhelmingly high proportion relative to the subcomponents when theantibiotic mixture is grossly separated.

The novel antibiotics of the present invention are amphoteric peptidesand thus are capable of forming acid-addition salts with a variety ofpharmacologically acceptable organic and inorganic salt formingreagents. Such salts may be prepared from the appropriate form of theantibiotic and one or more equivalents of an acid such as sulfuric,phosphoric, hydrochloric, hydrobromic, sulfamic, citric, maleic,fumaric, tartaric, acetic, benzoic, gluconic, ascorbic and the like. Thepharmacologically acceptable acid-addition salts would be expected to besolids, as is typical of amphoteric polypeptides in general. It followsthat they would be relatively soluble in water, methanol and ethanol butwould be relatively insoluble in non-polar organic solvents such asdiethyl ether, chloroform, benzene, toluene and the like.

Spectroscopic characteristics provided by ¹³ CNMR data will generallyserve to identify an organic compound being examined. The informationprovided will be characteristic of that organic compound alone.Moreover, the frequencies of the individual absorptions of the ¹³ CNMRcan be measured with high precision and will not be affected by theconcentration of the organic compound being examined. Therefore, eachset of significant carbon-13 nuclear magnetic resonance absorptions(Abs) produced by each of the antibiotics of the invention uniquelycharacterizes each and differentiates it from other polypeptideantibiotics. These sets of significant ¹³ CNMR absorptions forLL-BO2964α, β, and γ are given in Table I. The absorption (Abs) valuesare in parts per million in dimethyl sulfoxide-d₆ (DMSO-d₆) relative totetramethyl silane (TMS). The Peak Nos. correspond to the numbered peaksof the ¹³ CNMR spectra.

                  TABLE I                                                         ______________________________________                                        Sets Of Significant .sup.13 CNMR Absorptions (Abs)                            For the Primary Components LL-BO2964α, β and γ               Peak No.                                                                             Abs      Peak No. Abs     Peak No.                                                                             Abs                                   ______________________________________                                        α Primary Component                                                     LL-BO2964α                                                              1      13.9     16       96.7    31     137.3                                 2      18.4     17       102.1   32                                           3      27.3     18               33     138.9                                 4               19               34     139.6                                 5      29.3     20       112.9   35     140.4                                 6      30.5     21       113.4   36     150.3                                 7      33.9     22       116.2   37     152.9                                 8      48.5     23               38     156.9                                 9      49.5     24       119.3   39     157.3                                 10     50.4     25       119.9   40     162.7                                 11     55.0     26               41     166.4                                 12     69.9     27       127.6   42     170.1                                 13     71.4     28       128.6   43     171.0                                 14     73.0     29       129.6   44                                           15     91.5     30       135.8   45     173.1                                 β Primary Component                                                      LL-BO2964β                                                               1      13.9     16       96.7    31                                           2      18.3     17       102.1   32     138.4                                 3      27.3     18               33     138.9                                 4               19               34                                           5      29.3     20       113.4   35     140.4                                 6      30.4     21               36     150.3                                 7      33.9     22       115.7   37     152.9                                 8      48.9     23               38     156.9                                 9      49.6     24               39     157.3                                 10     50.4     25       119.4   40     162.7                                 11     54.9     26       125.7   41     166.4                                 12     69.4     27       127.7   42     169.9                                 13     71.3     28       129.3   43     171.0                                 14              29               44                                           15     91.4     30               45     173.1                                 γ Primary Component                                                     LL-BO2964γ                                                              1      13.9     16       96.7    31                                           2      18.4     17       102.2   32                                           3      27.4     18       110.3   33     138.9                                 4      28.1     19       111.0   34                                           5      29.3     20       113.4   35     140.4                                 6      30.4     21               36     150.3                                 7      33.9     22       115.7   37     153.0                                 8      48.7     23       118.0   38     156.9                                 9      49.4     24       118.5   39     157.4                                 10     50.4     25       120.5   40     162.7                                 11     54.2     26       123.4   41     166.4                                 12     69.5     27       127.6   42     170.0                                 13     71.4     28       129.0   43     171.1                                 14              29               44     171.9                                 15     91.5     30       135.8   45     173.1                                                                  46     174.9                                 ______________________________________                                    

The antibiotic components α, β and γ are isolated as noncrystalline,white fluffy powders. Their approximate elemental compositions are shownin Table II.

                  TABLE II                                                        ______________________________________                                        Elemental Analysis of LL-BO2964 Components                                    Component                                                                              % Carbon  % Hydrogen % Nitrogen                                                                            % Sulfur                                ______________________________________                                        LL-BO2964α                                                                       50.9      5.7        14.4    0                                       LL-BO2964β                                                                        50.9      5.8        13.6    0                                       LL-BO2964γ                                                                       52.9      5.8        15.1    0                                       ______________________________________                                    

The optical rotation values of the α, β and γ antibiotics are shown inTable III.

                  TABLE III                                                       ______________________________________                                        Optical Rotation of LL-BO2964 Components                                      Component    Optical Rotation                                                 ______________________________________                                        LL-BO2964α                                                                           [α].sub.D.sup.25 = -10° (C = 2.77, water)           LL-BO2964β                                                                            [α].sub.D.sup.25 = -19 ± 2° (C = 0.5,                         water)                                                           LL-BO2964γ                                                                           [α].sub.D.sup.25 = -20 ± 2° (C = 0.5,            ______________________________________                                                     water)                                                       

The UV-VIS, IR, ¹ HNMR and ¹³ CNMR spectra of the LL-BO2964 componentsare presented in FIGS. 1 through 15 and are correlated in foregoingChart A. The numbered peaks of the ¹³ CNMR spectra correspond to theappropriate antibiotic peak numbers listed in Table I.

The nucleotide base and amino acid residues produced by acid degradationof each of the antibiotics of the invention include uracil and thefollowing amino acids.______________________________________NH_(2CH)_(2COOH) glycine ##STR1## alanine NH_(2CH) _(2CH) _(2COOH) β-alanine##STR2## 2-amino-3-(methylamino)- butyric acid ##STR3##m-tyrosine______________________________________

Based on the amounts of each base or amino acid found, the polypeptidechain of each antibiotic of the invention at least contains one residueeach of uracil, glycine, alanine, β-alanine,2-amino-3-(methylamino)butyric acid and m-tyrosine.

In addition to uracil and the foregoing five amino acids, a hydantoin(A), formed from alanine and m-tyrosine, is present in the acidhydrolysate of LL-BO2964α, a hydantoin (B), formed from alanine andphenylalanine is present in the acid hydrolysate of LL-BO2964β, and ahydantoin (C), formed from alanine and p-tyrosine, is present in theacid hydrolysate of LL-BO2964γ. The meta and para substitutions of thetyrosines forming the A and C hydantoins are probable but not certain.These hydantoins have the followingformulas:______________________________________ ##STR4## Hydantoins Aand C ##STR5## Hydantoin B______________________________________

Chemical reaction studies of LL-BO2964γ have shown that it contains onlyone free amino group which can be N-acylated with acetic anhydride inmethanol. This group has been identified as the N-methylaminofunctionality of 2-amino-3-(methylamino)butyric acid. It is believedthat the α and β components would provide similar reaction results withacetic anhydride in methanol.

Although these degradation data do not permit elucidation of a molecularstructure for any of the α, β and γ antibiotic components, ahypothetical partial molecular structure of each component based onthese data is a five unit peptide chain of the foregoing five amino acidresidues arranged in an unknown sequence, which may be different foreach individual component, wherein each chain may be joined to a uracilmoiety at the carboxyl terminus. An intact hydantoin moiety having anidentity relating to each individual component as described above mayalso be present but it is more likely that the amino acids making up thehydantoin moiety are actually present in the molecular structure and thehydantoin is probably formed during hydrolysis. Other moieties such asamino sugars and the like may also be present. It will be understoodthat these hypothetical structures do not constitute any manner oflimitation of the invention.

Chemical comparison of the four A38533 antibiotic complex factorsdescribed in U.S. Pat. No. 4,180,564 and the LL-BO2964α, β and γantibiotic components of the present invention demonstrates theirstructural differences. For the four A38533 factors A1, A2, B and C, theelemental analyses reported for A1 and B, which are the major factors,indicate that they contain sulfur. The antibiotics of the invention, incontrast, have been analyzed for sulfur but do not contain it. Factor Cof the A38533 complex exhibits a UV chromophore having a maximumabsorption at 290 nm while the α, β and γ components of the inventionshow no similar UV absorption maximum. Finally, the retention time forhigh pressure liquid chromatography (HPLC) of the A38533 factors aresignificantly different from the HPLC retention times of the α, β and γcomponents of the invention when tested at the same time and under thesame conditions. The details of the HPLC comparison are given in TableX, infra.

The antibiotics of the invention are formed by the cultivation, undercontrolled conditions, of Streptomyces coeruleorubidus subspeciesrubidus. A representative strain of this microbe was isolated from asoil sample collected in Prescott, Ariz. and designated BO2964. Anultraviolet-induced mutant of this strain, designated BO2964 UV 14, hasalso been cultivated and produces antibiotics LL-BO2964α, LL-BO2964β,and LL-BO2964γ. They are both maintained in the culture collection ofthe Medical Research Division, American Cyanamid Company, Pearl River,N.Y. Viable cultures of both have been deposited with the CultureCollection Laboratory, Northern Utilization Research and DevelopmentDivision, U.S. Department of Agriculture, Peoria, Ill., under accessionnumbers NRRL 12372 and NRRL 12373 (mutant).

The following general description characterizes the microorganismsStreptomyces coeruleorubidus subspecies rubidus as represented by thestrain NRRL 12372. The UV mutant strain of Streptomyces coeruleorubidussubspecies rubidus as represented by NRRL 12373 has also beencharacterized and differences between the parent and mutant strains arepointed out where appropriate.

Observations of the cultural, physiological and morphological featuresof the microorganism were made in accordance with the methods detailedby Shirling, E. B. and D. Gottlieb, Internat. J. of Syst. Bacteriol. 16:313-340 (1966). Media used were selected from those recommended byPridham, et. al., Antibiotics Annual, 947-953 (1956/57) and Gordon, R.E., "The Taxonomy of Soil Bacteria", in The Ecology of Soil Bacteria,[T. G. R. Gray and D. Parkinson, eds.], pp. 293-321, LiverpoolUniversity Press, Liverpool, England (1967), for the taxonomic study ofactinomycetes and soil bacteria, respectively. Chemical composition ofthe cell walls was determined using the method of Lechevalier, et. al.,Adv. Appl. Microbiol. 14: 47-52 (1971) as modified by Stancek, J. L. andG. D. Roberts, Appl. Microbiol. 28: 226-234 (1974). Details aretabulated in the Examples section and overall descriptions of thecultures are summarized immediately hereafter. Underscored descriptivecolors are taken from Kelly, K. L. and D. B. Judd, "Color. UniversalLanguage and Dictionary of Names" , U.S. Nat. Bur. Stand. Spec. Publ.440, Washington, D.C. (1976) and the accompanying Inter-Society ColorCouncil, National Bureau of Standards Centroid Color Charts.

MICROMORPHOLOGY

Spores are formed in long spiral chains (spira) on aerial sporophores.The spores are ovoid (0.7-1.0 micron×0.9-1.0 micron) and the surface ofthe mature spores is ornamented with spines approximately 100 nanometersin length. Both strains appear similar when observed by scanningelectron microscopy.

CELL WALL COMPOSITION

Whole-Cell hydrolysates of these cultures contain the L,L-isomer ofdiaminopimelic acid, placing them in the Type I cell wall groupclassified according to the method of Lechevalier, cited above, asmodified by Stancek, cited above. This is typical of all Streptomycesspecies.

AMOUNT OF GROWTH

Good growth is observed on most media; moderate growth is observed oninorganic salts-starch agar and nutrient agar.

AERIAL MYCELIUM AND SPORE COLOR

Aerial mycelium is white; spore masses are blue shades ranging from 185,pale blue to 190. light bluish gray. Sporulation is moderate to heavy,dependent on the medium. The mutant strain generally exhibits lesssporulation than the parent strain on most media.

SOLUBLE PIGMENTS

Soluble pigments are absent when the strains are grown on nutrient mediaincluding Benedict's agar, glycerol-aspargine agar and oatmeal agar.Reddish to reddish-brown pigments are produced by both strains whengrown on the following nutrient media: asparagine-dextrose agar,Czapeck's agar, yeast extract-malt extract agar, Bennett's agar,Hickey-Tresner agar, inorganic salts starch agar, tomato paste-oatmealagar. Pigment production is somewhat suppressed in the mutant strain.

REVERSE COLOR

The reverse color produced by the strains grown on the nutrient mediaproducing soluble pigment are pinkish-yellow to reddish-brown shades.

PHYSIOLOGICAL REACTIONS

The cultures display the following physiological reactions: nitratesreduced to nitrites; partial liquefaction of gelatin in 14 days;melanoid pigments produced on both peptone-yeast extract agar andtyrosine medium; hydrolysis of adenine, hypoxanthine and tyrosine, butnot guanine and xanthine in 7 days. Carbohydrate utilization as per themethod of Pridham, T. G. and D. Gottlieb, J. Bacteriol. 56: 107-114(1948): good utilization of galactose, glucose, maltose, mannose,melibiose and xylose; moderate utilization of adonitol, arabinose,fructose, inositol, lactose, mannitol, raffinose, rhamnose, ribose andtrehalose; poor utilization of sucrose; no utilization of glycerol ormelezitose. The mutant strain exhibits moderate utilization of sucrosebut poor utilization of melibiose. Both the parent and mutant strainstolerate 7% but not 10% sodium chloride.

A culture of the parent strain has been compared with Streptomycesreference cultures which have similar characteristics including bluespores, chromogenicity, and spiral spore chains with spiny spores. Ofthe four characteristic observed, which include spore color, solublepigment, reverse color and amount of growth, no reference Streptomycesreference culture exhibited the same four characteristics as the S.coeruleorubidus rubidus of the invention. These comparisons arepresented in detail in the Examples section.

Characterization of the microbial cultures of the invention as S.coeruleorubidus rubidus has been made because they most closely resemblethe reference strain Streptomyces coeruleorubidus. The vegetativemycelia of the cultures on most media tend to have yellowish-pink shadesand the cultures produce red soluble pigments on defined media.Moreover, the physiological reactions of the cultures also closelyresemble those of the reference strain, although the reference straindoes not reduce nitrates. The cultures of the invention are thereforeassigned to the taxon Streptomyces coeruleorubidus, but because theirproduction of red soluble pigments is far more prolific than thereference strain, they have been named as a new subspecies, namelyStreptomyces coeruleorubidus subspecies rubidus.

It will be understood that for the purposes of the present invention,the production of the new antibiotics, LL-BO2964α, β, and γ, is notlimited to the S. coeruleorubidus rubidus organism described in theforegoing passage or to microbes fitting the foregoing morphological,physiological growth characteristics. Use of these organisms is givenfor illustrative purposes only and production by any microbial means orcombination of microbial and synthetic means is applicable to theinvention. In fact, it is desired and intended to include the use ofnatural (spontaneous) mutants of this organism as well as induced andgenetically engineered mutants produced from this organism by variousmeans such as exposure to X-ray irradiation, ultraviolet irradiation,nitrogen mustard, actinophages, gene manipulation, recombinant DNAtechniques and the like.

The antibiotics of the invention are active in vitro against grampositive and gram negative bacteria as shown by the minimal inhibitoryconcentrations of the antibiotics (MIC values) in mcg./ml. summarized inTable IV. The MIC's were determined by standard agar dilution proceduresknwon to those skilled in the art. The MIC observations were made afterincubation for 18 hours at 35° C. on Mueller-Hinton agar.

                                      TABLE IV                                    __________________________________________________________________________    IN VITRO ANTIBACTERIAL ACTIVITY OF THE                                        ANTIBIOTICS OF THE INVENTION                                                                   Minimal Inhibitory                                                            Concentration (mcg/ml) (e)                                                    Cell                                                                             LL-BO2964                                                                             LL-BO2964                                                          Type                                                                             Mixture of                                                                            Mixture of                                                                            LL-BO2964γ                          Organism         (a)                                                                              α, β and γ (b)                                                       α and β (c)                                                                (d)                                       __________________________________________________________________________    Pseudomonas aeruginosa SSC-78-13                                                               -    64      64      64                                      Pseudomonas aeruginosa Strain 1                                                                     16      16      16                                      Pseudomonas aeruginosa Strain 2                                                                     16      16      16                                      Pseudomonas aeruginosa Strain 3                                                                     16      16      16                                      Pseudomonas aeruginosa Strain 4                                                                   >1024   >1024    1024                                     Klebsiella pneumoniae AD                                                                       -    128     256     64                                      Enterococcus OSU-75-1                                                                          +  >1024    1024     256                                     SM-77-15            >1024     512     256                                     Micrococcus lutea PCI 1001                                                                     +    256     256     256                                     Serratia marcescens  TUL-78-15                                                                 -  >1024   >1024   >1024                                     Salmonella Sp. SSC-79-57                                                                       -  >1024   >1024   >1024                                     Escherichia coli #331                                                                          -  >1024   >1024   >1024                                     Enterobacter aerogenes STFD-79-14                                                              -  >1024   >1024   >1024                                     __________________________________________________________________________     (a) Cell type: gram positive is designated + and gram negative is             designated -.                                                                 (b) This LLBO2964 mixture of α, β and γ components           contained 4% α, 34% β and 62% γ according to high            pressure liquid chromatographic analysis.                                     (c) This LLBO2964 mixture of α and β components contained 40%      α and 60% β according to high pressure liquid chromatographic      analysis.                                                                     (d) The LLBO2964γ used was substantially pure and contained about       90% γ component, 0% α component and about 10% unidentified        impurities according to high pressure liquid chromatographic analysis.        (e) The MIC value of >1024 means the MIC value is above the highest           concentration of antibiotics tested (1024 mcg/ml).                       

Antibiotics LL-B02964α, β and γ are also active in vivo againstbacterial infections as demonstrated by their ability to controlPseudomonas, Klebsiella and Staphylococcus infections in mice. Forexample, the LL-B02964 mixture of α, β and γ components shows in vivoantibacterial activity against Pseudomonas aeruginosa 12-4-4 whenadministered to mice infected with this bacterium. In the test, theanimals used were Charles River Lab CD-1 mice each weighing 20±2 g. Themice were injected intraperitoneally with 0.5 ml of P. aeruginosa[1.2×10⁵ CF units of microbe] contained in 5% mucin. LL-B02964antibiotic treatment doses were contained in 0.5 ml of 0.2% aqueous agarand were administered subcutaneously at 0, 2 and 4 hours afterinoculation. Table V summarizes the in vivo activity of the LL-B02964antibiotic in this test. The results are recorded as number of micealive over the total mice tested 7 days after infection.

                  TABLE V                                                         ______________________________________                                        IN VIVO ACTIVITY OF LL-BO2964(a) AGAINST                                      P. aeruginosa                                                                 LL-BO2964(a)                                                                  Dosage Amounts Alive/Total Mice Tested                                        In mg/kg Per Dose                                                                            7 Days After Infection                                         ______________________________________                                        1024           5/5                                                             512           4/5                                                            Untreated       0/10                                                          controls                                                                      ______________________________________                                         (a)The LLBO2964 antibiotic mixture used in this in vivo test contains 18%     α, 30% β and 52% γ components according to high pressure     liquid chromatographic analysis.                                         

Tests with Klebsiella and Staphylococcus showed approximately the sameactivity. In addition to their spectrum of antibacterial activity, theantibiotics of the invention are effective against Mycobacteriumsmegmatis and and Mycobacterium tuberculosis. In particular, antibioticLL-B02964 containing a mixture of the α, β and γ components, antibioticLL-B02964 containing the α and β components, and antibiotic LL-B02964γwere tested for bactericidal activity against Mycobacterium smegmatisATCC 607 in Sauton's medium modified with 0.1% polysorbate 80. The testwas conducted according to the procedure of A. L. Barry and L. D.Sabath, "Manual of Clinical Microbiology", 2nd Edition, p. 431 (1974).The results are summarized in Table VI.

                  TABLE VI                                                        ______________________________________                                        ACTIVITY OF THE ANTIBIOTICS AGAINST                                           M. SMEGMATIS                                                                            Colony Forming Units/ml. After                                      Concentration                                                                           20 Hours Incubation at 37° C. (c)                            of antibiotic                                                                           LL-BO2964 (a)                                                                             LL-BO2964 (b)                                           tested in α, β and γ                                                               α and β                                      mcg/ml    components  components  LL-BO2964γ                            ______________________________________                                        256       1.2 × 10.sup.3                                                                      8.2 × 10.sup.3                                                                      4.1 × 10.sup.3                        128       1.0 × 10.sup.3                                                                      2.3 × 10.sup.3                                                                      1.4 × 10.sup.3                         64       9.7 × 10.sup.5                                                                      5.6 × 10.sup.4                                                                      3.2 × 10.sup.6                        ______________________________________                                         (a) This LLBO2964 mixture contained 4% α, 34% β and 62%            γ.                                                                      (b) This LLBO2964 mixture contained 40% α and 60% β.               (c) Inoculum: 8 × 10.sup.6 colony forming units/ml.                

Using the following procedure, antibiotic LL-B02964 containing α, β andγ components and antibiotics LL-B02964β and γ were tested for activityagainst Mycobacterium tuberculosis H37Rv. Test groups of five CharlesRiver CF-1 female albino mice, each weighing about 18-20 g., wereintravenously injected with 0.2 ml. of a buffered saline suspensioncontaining 0.3 mg. (wet weight) of cells gathered from a 14-day oldculture of Mycobacterium tuberculosis H37Rv, grown on Sauton's agar. Theantibiotics were daily administered by gavage (o.t.), subcutaneous(s.c.) or intraperitoneal (i.p.) routes to the test groups of mice andeach test group received a different dose. The antibiotic formulationsfor administration constituted suspensions in 0.2% aqueous agar orphysiological saline with the concentrations of the antibiotics adjustedso that the required daily dose volume was 0.5 ml. The course oftreatment started with the day of infection and ended 14 days later.Antibiotic activity was measured by the survival ratios of mice at 30days postinfection. Deaths were recorded daily. Antibiotics protectingat least two out of five mice for 30 days were considered to be active.The positive and null control groups for the tests conducted on eachantibiotic constituted ten to twenty infected, untreated mice (IUC) andten unifected, untreated mice (UUC). Only one of the 70 infected miceused as the positive controls (IUC) for the total number of testssurvived the 30 day test period. The results of the tests are summarizedin Tables VII through VIII. Tests using differing routes ofadministration of LL-B02964 were conducted as separate studies and arenoted as runs 1 and 2 in Table VII.

                  TABLE VII                                                       ______________________________________                                        ANTITUBERCULER ACTIVITY OF LL-BO2964                                          (α, β and γ components)*                                     Dose of LL-BO2964                                                             (α, β, γ)*                                                   in mg./kg./day                                                                             30-Day Postinfection Survival Ratios                             ______________________________________                                                   s.c.       o.t.                                                               Run 1                                                              ______________________________________                                        400          5/5          --                                                  200          --           4/5                                                 100          2/5          3/5, 5/5.sup.+                                       50          --           3/5                                                 Infected, untreated                                                                         0/10         1/20                                               controls (IUC)                                                                ______________________________________                                                     i.p.         o.t.                                                           Run 2                                                              ______________________________________                                        400          2/5          --                                                  100          3/5          3/5                                                 IUC**        0/20                                                             ______________________________________                                         *The weight percent of components in this LLBO2964 mixture was 4% α     34% β and 62% γ.                                                   .sup.+ In a separate study, a second group was tested O.T. at this dosage     and had the indicated survival ratio.                                         **One IUC group of 20 mice was used for Run 2.                           

                  TABLE VIII                                                      ______________________________________                                        ANTITUBERCULAR ACTIVITY OF LL-BO2964β and                                LL-BO2964γ                                                              Gavage (o.t.) dose                                                            of β or γ                                                                       30-Day Postinfection Survival Ratios                             in mg./kg./day                                                                             LL-BO2964β                                                                            LL-BO2964γ                                    ______________________________________                                        400          4/5          3/5                                                 IUC          0/20                                                             ______________________________________                                    

Cultivation or fermentation of Streptomyces coeruleorubidus subspeciesrubidus NRRL 12372 or NRRL 12373 may be carried out in a wide variety ofliquid media. Media which will be useful include those containing anassimilable source of carbon such as starch, sugar, molasses, and thelike; an assimilable source of nitrogen such as protein, proteinhydrolysate, polypeptides, amino acids, corn steep liquor and the like;and inorganic anions and cations known to be necessary for microbialgrowth, such as potassium, sodium, ammonium, calcium, sulfate,carbonate, phosphate, chloride and the like. Trace elements which arealso known to be necessary for microbial growth, such as boron,molybdenum, copper and the like will be supplied as impurities of otherconstituents of the media. Aeration in tanks, bottles and flasks can beprovided by forcing sterile air through the fermenting medium or ontoits surface. Further agitation in tanks can be provided by mechanicalmeans. Anti-foaming agents such as lard oil or silicone defoamer may beadded where appropriate. Specific methods and procedures forfermentation are provided in the Examples section.

Generally, the antibiotics LL-B02964α, β and γ can be isolated andpurified by methods known to those skilled in the art. These includefiltration through filter aids such as diatomaceous earth and the like,chromatography on molecular gel or ion exchange columsn, high pressureliquid chromatography, electrophoresis, dialysis, electrodialysis andpartition between organic and aqueous solvents. In a typical procedure,the LL-B02964α, β and γ components can be isolated and grossly separatedfrom the fermentation broth by absorption of the fermentation filtrateon a column of macroreticular resin and elution of the column withaqueous alcohol mixtures. The LL-B02964 component mixture can bepartitioned between 1-butanol and water and the antibiotic components,which will be contained in the 1-butanol phase, can be concentrated byvacuum removal of the solvent and further purified by chromatography ona cation exchange molecular gel such as CM-SEPHADEX® [a modified dextrincation exchange gel manufactured by Pharmacia, Inc., Piscataway, N.J.].The substantial separation of the α,β and γ antibiotic components can beachieved by molecular gel filtration chromatography or partitionchromatography on such materials as cross-linked, swelled dextrin gel,microcrystalline cellulose, or polyacetylamide gels.

Substantial separation of the individual α, β and γ antibioticcomponents as well as identification of the components separatedaccording to the foregoing procedures can be respectively andconveniently accomplished by preparative or analytical high pressureliquid chromatography (HPLC). Generally, those skilled in the art willbe able to ascertain column and eluant types to produce satisfactoryresults. Columns such as gels, macroreticular resins and polyacrylamideand aqueous solutions of polar organic solvents, in particular, will beuseful. The preferred HPLC columns for the separation and identificationof the α, β and γ antibiotic components are a column of Zorbax ODS® [anabsorptive bead material manufactured by Dupont, Inc., Wilmington,Del.]; or Ultrasphere-Octyl® [a coating silica manufactured by Altex,Inc., California]. Typical HPLC conditions employed with these columnsare a solvent system containing 0.1M ammonium acetate at pH 5.0 andacetonitrile at a ratio of 85/15 parts by volume; a flow rate of 1.2 mlper minute; and a UV variable detector at 254 nm and 275 nm wavelenghts.Other conditions will be readily determinable by those skilled in theart.

The antibiotic component retention times and volumes on a Zorbax ODS®column under the foregoing typical conditions, which were produced byintroduction of 2.5 micrograms α, 5 micrograms β and 5 micrograms γ, aregiven in Table IX.

                  TABLE IX                                                        ______________________________________                                        HPLC OF ANTIBIOTICS ON ZORBAX ODS ®                                       Component                                                                             Retention Time (minutes)                                                                       Retention Volume (ml.)                               ______________________________________                                        α 3.1              3.7                                                  β  5.5              6.6                                                  γ 7.7              9.2                                                  ______________________________________                                    

A comparative study of the HPLC curves produced by the antibioticcomponents of the invention and the factors of the A38533 complexdescribed in U.S. Pat. No. 4,180,564 was conducted under the sameconditions except that a column of Ultrasphere-Octyl® was used. Theresults of this study were recorded as retention volumes (RV) in ml. andare presented in Table X.

                  TABLE X                                                         ______________________________________                                        COMPARATIVE STUDY ON                                                          ULTRASPHERE-OCTYL ®                                                       A38533 Factor                                                                           RV (ml)   LL-BO2964 component                                                                           RV (ml)                                   ______________________________________                                        A1         7.2      α         5.1                                       A2        19.2      β          9.0                                       B         34.8      γ         13.8                                      C         56.4                                                                ______________________________________                                    

The following Examples further describe several embodiments of theinvention including morphological, physiological and growthcharacteristics of cultures of S. coeruleorubidus rubidus on severaltypes of media, methods for the fermentation of this microbe and methodsfor the production of the α,β and γ antibiotic components. Theseexamples are not meant to be limitations of the invention, however.

EXAMPLE 1 Culture Characterization and Fermentation of Streptomycescoeruleorubidus rubidus

A. Culture Characterization

Observations of the cultural, physiological and morphological featuresof S. coeruleorubidus rubidus were made in accordance with the methodsdetailed by E. G. Shirling, and D. Gottlieb, cited supra. The culturemedia used were selected from those recommended by Pridham and P. E.Gordon for the taxonomic study of actinomycetes and soil bacteria, citedsupra. Chemical composition of the cell walls was determined using themethod of Lechevalier, as modified by J. L. Stancek, cited supra.Underscored descriptive colors are taken from K. L. Kelly and D. B.Judd, "Color. Universal Language and Dictionary of Names", cited supra,and the accompanying Inter-Society Color Council, National Bureau ofStandards Centroid Color Charts.

Tables XI, XII and XIII, which follow, respectively summarize theobserved cultural characteristics of the microorganism of the inventionin standard media, miscellaneous physiological reactions and carbonsource utilization. The cultural, physiological and utilizationdifferences exhibited by mutant strain NRRL 12373 are noted in theoverall description of these characteristics given in the foregoinggeneral discussion of the microbes of the invention.

                                      TABLE XI                                    __________________________________________________________________________    CULTURAL CHARACTERISTICS OF Streptomyces coeruleorubidus subsp.               rubidus NRRL 12372 and NRRL 12373*                                            Incubation period: 14 days at 28° C.                                              AMOUNT                                                                        OF    AERIAL MYCELIUM                                                                           SOLUBLE                                                                             REVERSE                                    MEDIUM     GROWTH                                                                              and/or SPORES                                                                             PIGMENT                                                                             COLOR                                      __________________________________________________________________________    Asparagine-Dextrose                                                                      good  white aerial mycelia                                                                      reddish                                                                             30. dark                                                    becoming 190. light                                                                             yellowish                                                   bluish gray in sporu-                                                                           pink                                                        lating areas. Sporu-                                                          lation heavy.                                                Bennett's Agar                                                                           good  white aerial mycelia                                                                      reddish-                                                                            30. dark                                                    becoming 190. light                                                                       brown yellowish                                                   bluish gray in sporu-                                                                           pink                                                        lating areas. Sporu-                                                          lation heavy.                                                Benedict's Agar                                                                          good  trace of white aerial                                                                     none  73. pale                                                    mycelia; no sporula-                                                                            orange                                                      tion              yellow                                     Czapek's Agar                                                                            good  white aerial mycelia;                                                                     reddish                                                                             29. moderate                                                no sporulation.   yellowish                                                                     pink                                       Glycerol-Asparagine                                                                      good  white aerial mycelia                                                                      none  29. moderate                                                becoming 190. light                                                                             yellowish                                                   bluish gray to 185.                                                                             pink                                                        pale blue in sporula-                                                         tion areas. Sporula-                                                          tion heavy.                                                  Hickey-Tresner                                                                           good  white aerial mycelia                                                                      reddish-                                                                            47. dark                                   Agar             becoming 190. light                                                                       brown grayish                                                     bluish gray in sporu-                                                                           reddish                                                     lated areas. Sporula-                                                                           brown                                                       tion heavy.                                                  Inorganic Salts                                                                          moderate                                                                            sparse white aerial                                                                       faint 31. pale                                   Starch Agar      mycelia; no sporu-                                                                        reddish                                                                             yellowish                                                   lation.           pink                                       Nutrient Agar                                                                            moderate                                                                            white aerial mycelia;                                                                     brownish                                                                            33. brownish                                                no sporulation.   pink                                       Oatmeal Agar                                                                             good  white to yellowish                                                                        none  colorless                                                   aerial mycelia be-                                                            coming 190. light blu-                                                        ish gray to 185. pale                                                         blue in sporulated                                                            areas. sporulation                                                            heavy                                                        Tomato-Paste-                                                                            good  white to yellowish                                                                        reddish-                                                                            47. dark                                   Oatmeal Agar     aerial mycelia be-                                                                        brown grayish                                                     coming 190. light reddish                                                     bluish gray; sporu-                                                                             brown                                                       lation heavy                                                 Yeast extract                                                                            good  white aerial mycelia                                                                      reddish-                                                                            47. dark                                                    becoming 190. light                                                                       brown grayish                                                     bluish gray in sporu-                                                                           reddish                                                     lated areas. sporula-                                                                           brown                                                       tion heavy                                                   __________________________________________________________________________     *NRRL 12373 differences as noted in general discussion of the microbes.  

                  TABLE XII                                                       ______________________________________                                        MISCELLANEOUS PHYSIOLOGICAL REACTIONS OF                                      Streptomyces coeruleorubidus subsp. rubidus NRRL 12373 and                    NRRL 12373*                                                                              INCU-     AMOUNT                                                              BATION    OF        PHYSIOLOGICAL                                  MEDIUM     PERIOD    GROWTH    REACTION                                       ______________________________________                                        Peptone-Iron                                                                             72    hours   good    moderate amount of                           Agar                             melanoid pigment                                                              produced                                     Tyrosine Medium                                                                          3     days    good    slight blackening                                       7     days    good    slight blackening                            Litmus Milk                                                                              7     days    good    slight proteolysis                                      14    days    good    neutral pH; moder-                                                            ate peptonization                            Nutrient Gelatin                                                                         7     days    good    slight proteolysis                                      14    days    good    moderate proteoly-                                                            sis                                          Organic Nitrate                                                                          7     days    good    nitrates reduced                             Broth                            to nitrites                                             14    days    good    nitrates reduced                                                              to nitrites                                  NaCl Tolerance                                                                           14    days            tolerates 7% NaCl                            Agar                             but not 10% NaCl                             Adenine Agar                                                                             7     days    good    hydrolysis                                   Guanine Agar                                                                             7     days    good    hydrolysis                                   Hypoxanthine                                                                             7     days    good    hydrolysis; mela-                            Agar                             noid pigment pro-                                                             duction                                      Tyrosine Agar                                                                            7     days    good    hydrolysis; mela-                                                             noid pigment pro-                                                             duction                                      Xanthine   7     days    good    no hydrolysis                                ______________________________________                                         *NRRL 12373 differences as noted in the general discussion of the             microbes.                                                                

                  TABLE XIII                                                      ______________________________________                                        CARBON SOURCE UTILIZATION PATTERN OF                                          STREPTOMYCES COERULEORUBIDUS SUBSPECIES                                       RUBIDUS NRRL 12373 and NRRL 12373*                                            Incubation period: 14 days at 28° C.                                   CARBON SOURCE    UTILIZATION.sup.+                                            ______________________________________                                        Adonitol         2                                                            1-Arabinose      2                                                            Fructose         2                                                            d-Galactose      3                                                            d-Glucose        3                                                            Glycerol         0                                                            i-Inositol       2                                                            Lactose          2                                                            Maltose          3                                                            d-Mannitol       2                                                            d-Mannose        3                                                            d-Melezitose     0                                                            d-Melibiose      3                                                            d-Raffinose      2                                                            1-Rhamnose       2                                                            d-Ribose         2                                                            Sucrose          1                                                            d-Trehalose      2                                                            d-Xylose         3                                                            Negative Control 0                                                            ______________________________________                                         *NRRL 12373 differences as noted in the general discussion of the microbe     .sup.+ 3 = Good utilization                                                   2 = fair utilization                                                          1 = Poor utilization                                                          0 = No utilization                                                       

Comparative studies of the morphological and growth characteristics ofthe parent strain NRRL 12372 and several reference strains ofStreptomyces microorganisms were conducted by culturing slants of thestrains on asparagine-dextrose agar at about 38° C. for about 14 daysaccording to standard culture production methods. The comparative sporecolor, soluble pigment, reverse color and growth characteristics ofthese strains observed during these studies are summarized in Table XIV.

                                      TABLE XIV                                   __________________________________________________________________________    COMPARATIVE OBSERVATIONS OF STREPTOMYCES CULTURES                             GROWN ON ASPARAGINE-DEXTROSE AGAR                                                                  SOLUBLE                                                  CULTURE     SPORE COLOR                                                                            PIGMENT                                                                             REVERSE COLOR                                                                            GROWTH                                  __________________________________________________________________________    S. caelestis                                                                              pale blue                                                                              yellow                                                                              light yellow                                                                             good                                    AZ801 (NRRL 2418)                                                             S. chartreusis                                                                            light bluish-                                                                          pale  light yellow                                                                             good                                    AE4 (NRRL B-2287)                                                                         gray to pale                                                                           yellow                                                               blue                                                              S. chartreusis                                                                            light    none  light      good                                    BE821 (NRRL B-2199)                                                                       greenish-gray  yellowish-                                                                    brown                                              S. coeruleofuscus                                                                         light    none  light to   good                                    BB102 (Gause 5051/56)                                                                     bluish-gray    moderate                                                                      yellow                                             S. coeruleorubidus                                                                        very pale                                                                              reddish                                                                             moderate   moderate                                BB103 (ATCC 13740)                                                                        blue           yellowish-                                                                    pink                                               S. coerulescens                                                                           light    none  pale yellow                                                                              moderate                                BB104 (Gause 4562)                                                                        bluish-gray                                                       S. coeruleorubidus                                                                        very pale                                                                              reddish                                                                             dark       good                                    rubidus     blue           yellowish-                                         (NRRL 12372)               pink                                               __________________________________________________________________________

B. Inoculum Preparation of S. coeruleorubidus rubidus

An inoculum medium having the following formulation wasprepared:______________________________________Cornstarch 24 g.Glucose 5g.Yeast extract 5 g.tryptone¹ 5 g.Beef extract 3 g.Calcium carbonate 4g.Water to 1000 ml.______________________________________ ¹ [A peptone,registered trademark of Difco Laboratories, Detroit, Michigan]

Washed or scraped spores from an agar slant of Streptomycescoeruleorubidus subspecies rubidus NRRL 12372 were used to inoculate 100ml. of the foregoing sterile medium contained in a 500 ml flask. Twosuch flasks were placed on a rotary shaker and agitated at 180 r.p.m.and 28° C. for 48 hours. The resulting flask inoculum was thentransferred to a 12 liter bottle containing the same sterile medium andincubated at 28° C. for 48 hours with sterile aeration. The resultingbottle inoculum was used to inoculate a tank containing 300 liters ofthe same sterile medium. The inoculated tank medium was incubated byagitating and aerating with sterile air at 28° C. for 24 hours toproduce a tank inoculum. The inoculation conditions for flasks, bottleand tank fermentation may also vary from 24°-35° C. and from 20-56hours. Aeration may be surface or in depth delivery of essential gases,typically sterile air through fritted glass, a bubble or other similardevice. A medium of cornstarch, maltose and soy peptone, sterilized andadjusted to pH 7 with phosphate or similar buffer may also be used.Silicone antifoaming agent or other similar agent may be added tocontrol fermentation frothing.

C. Bulk Fermentation

A fermentation medium of the following formula which was suitable forbulk production of S. coeruleorubidus rubidus wasprepared.______________________________________Cornstarch 10 g.Maltose20 g.Soy peptone 10 g.Water to 1000ml.______________________________________

Fifteen hundred liters of this medium adjusted to pH 7 was sterilized at120° C. for 60 minutes, and then inoculated with 150 liters of the tankS. coeruleorubidus rubidus inoculum described in section A. Bulkfermentation was then carried out at 28° C., using a silicone antifoamto control frothing. Aeration was supplied as 0.66 liters of sterile airper liter of microbe-medium mash per minute. The microbe-medium mash wasagitated by impellers driven at 100 r.p.m. After fermenting forapproximately 90 hours, the mash was harvested.

EXAMPLE 2 Preparation Of The α, β And γ Antibiotic Components

A. Isolation of The Components

A fermentation is carried out as described in Example 1. The harvestmash (1450 l.) produced by the bulk fermentation of Example 1 wasadjusted to pH 5.5 with 1.2 l. of concentrated hydrochloric acid, thencombined with 43.5 kg. of diatomaceous earth and filtered through afilter press. The pad on the press was washed with water and the waterwash was pooled with the filtrate. The pooled filtrate (1500 l.) waspercolated through a 38 l. bed volume of cross-linked polystyrenemacroreticular resin Amberlite® XAD-2 [Rohm and Haas, Philadelphia,Pa.], at a flow rate of 1600-1900 ml. per minute. After percolation wascompleted, the resin was washed with 152 l. of demineralized water andthen eluted with 200 l. of methanol:water (4:1). Ten 20 l. fractionswere collected. Each fraction was checked for antibacterial activity bybioassay against E. coli or P. aeruginosa. Fractions 2-5 showed activityand were pooled and concentrated under reduced pressure to produce anantibiotic concentration in aqueous solution. This concentrate wasextracted with an equal volume of 1-butanol and the 1-butanol phaseseparated and saved. The aqueous phase was then adjusted to pH 2.9-3.1with 6N hydrochloric acid and extracted three times with equal volumesof 1-butanol. All butanol phases from both extractions were combined andconcentrated under reduced pressure to a volume of about 15 liters. Theresulting liquid concentrate was decanted and the remaining tarryresidue saved. The concentrate was centrifuged to cause sedimentationand both the supernatant and sedimented product collected and saved. Thesediment product was slurried with ethyl acetate and filtered. Thefiltered solid product was vacuum dried to yield 166 g. of yellowmaterial. The tarry residue from the still was washed with 4 liters of a1-butanol solution saturated with water. The saturated butanol washingwas pooled with the supernatant from the centrifugation and concentratedunder reduced pressure to about 3 liters which was cooled to causeprecipitation. The resulting precipitate is collected by centrifugation,washed with ethyl acetate, filtered and vacuum dried to yield anadditional 95 g. of solid product.

B. Purification of the α, β and γ Antibiotic Components

A chromatographic column (4.2×130 cm) was prepared with SP-Sephadex® gel(Pharmacia Fine Chemicals, Piscataway, N.J.). A 13 g. portion of theisolated α, β and γ antibiotic component mixture of preceeding section Awas added to 40 ml. of water, the pH adjusted to 7.8 using 1N sodiumhydroxide and the mixture gently warmed over steam to cause dissolution.The solution was then absorbed onto the column of Sephadex® gel and thecolumn was developed with water. Fractions of 45 ml. were collected andeach was checked for antibacterial activity by bioassay on large agarplates seeded with a strain of Escherichia coli and /or Pseudomonasaeruginosa. Fractions 24-89 were combined and freeze-dried to yieldabout 3.5 g. of the LL-B02964 mixture of α, β and γ antibioticcomponents.

In a second, alternative purification procedure, a chromatographiccolumn (4.2×130 cm.) was prepared with a gel of SP-Sephadex®. A 12 g.portion of the isolated α, β and γ component mixture of section A wasadded to 40 ml. of water, the resulting mixture gently warmed and the pHadjusted to 7.5-8.0 with 5N sodium hydroxide. This solution was thenabsorbed onto the Sephadex® gel column and the column washed with 25 ml.of water, then developed with 0.1M aqueous ammonium acetate. Fractionsof 45 ml. were collected and each was checked for antibacterial activityby the bioassay given supra. Fractions 21-35 were combined, desaltedusing Amberlite® XAD-2 resin and freeze-dried to yield about 7.7 g. ofthe LL-B02964 mixture of α, β and γ antibiotic components.

In general, the individual fractions produced by these purificationprocedures will contain a mixture of α, β and γ antibiotic componentswherein the proportional ratios of components will vary from fraction tofraction.

EXAMPLE 3 Separation Of Antibiotic LL-B02964γ And A Mixture OfAntibiotics LL-B02964α And β

A chromatographic column (5×90) was prepared with a polyacrylamide gel,Bio-Cel® P-4 (50-100 mesh) [Bio Rad Laboratories, Richmond, Calif.]). ALL-B02964 mixture of α, β and γ antibiotic components purified asdescribed in Example 2, and weighing about 2.4 g. was added to 40 ml. ofwater, the mixture warmed and the pH adjusted to 7.7 with diluteammonium hydroxide. The resulting solution was absorbed onto theBio-Gel® column, the column developed with water and fractions of 22 ml.collected. Aliquots of the column fractions were examined by thin layerchromatography on UV active polyamide plates. The chromatograms weredeveloped with 2M ammonium acetate-methanol (7:3 v/v) and the zones weredetected as yellow spots by spraying with a 1% aqueous solution ofp-nitrobenzenediazonium tetrafluoroborate. The antibiotics appeared asyellow zones on the thin layer chromatogram. Two antibiotic componentswere evident; the first, LL-B02964γ, had an Rf value of 0.65 and thesecond was composed of a mixture of LL-B02964α and LL-B02964β asidentified by analytical HPLC described supra.

Fractions 75-88, containing LL-B02964γ were combined and freeze-dried toyield 675 mg. of the γ compound (portion H). Fractions 64-68, containinga mixture of LL-B02964α and β were combined and freeze-dried to yield771 mg. of a mixture of the α and β components (portion I). Fractions69-74, containing all three components, α, β and γ, were combined andfreeze-dried, to yield 287 mg. of a mixture of the α, β and γ components(portion J).

LL-B02964γ isolated and separated according to this procedure had thefollowing characteristics:

Microanalysis (percent): C, 52.87; H, 5.75; N, 15.10; O (direct), 21.19;S, 0.00.

Optical Rotation: [α]_(D) ²⁶ =-20±2° (C=0.5%, water).

LL-B02964γ produced an infrared absorption spectrum (KBr pellet) asshown in FIG. 11; a U.V. spectrum in water exhibiting absorption maximaat 225 and 259 nm as shown in FIG. 12; ¹ HMR spectra relative totetramethyl silane in δppm, taken as a solution in D₆ dimethylsulfoxide, as shown in FIG. 13 and under the same conditions and withadded deutero trifluoroacetic acid as shown in FIG. 14; and a carbon-13NMR spectrum relative to tetramethyl silane in ppm, taken as a solutionin D₆ dimethyl sulfoxide, as shown in FIG. 15. The significant carbon-13NMR absorptions are tabularized in Table I.

EXAMPLE 4 Separation Of Antibiotic LL-B02964α

A chromatographic (5×90 cm) column was prepared with a gel ofpolyacrylamide gel Bio-Gel® P-4 (50-100 mesh). A 1682 mg. portion of amixture of α, β and γ antibiotic components similar to portion (J) ofExample 3 was dissolved in 40 ml. of water and absorbed onto the BioGel® column. The column was developed with water and 15 ml. fractionswere collected. Fractions 73-77, which contained LL-B02964 according toan HPLC analysis conducted as described supra, were combined andfreeze-dried, to yield 430 mg. of the antibiotic LL-B02964 having thefollowing characteristics:

Microanalysis (percent): C, 50.89; H, 5.67; N, 14.44; S, 0.00.

Optical Rotation: [α]_(D) ²⁵ =-10° (C=2.77%, water).

LL-B02964α produced an infrared absorption spectrum (KBr pellet) asshown in FIG. 1; a U.V. spectrum in water exhibiting absorption maximaat 221 and 254 nm as shown in FIG. 2; ¹ HMR spectra relative totetramethyl silane in ppm, taken as a solution in D₆ dimethyl sulfoxide,as shown in FIG. 3 and under the same conditions with added deuterotrifluoro acetic acid as shown in FIG. 4; and a carbon-13 NMR spectrumrelative to tetramethyl silane in δppm, taken as a solution in D₆dimethyl sulfoxide, as shown in FIG. 5. The significant carbon-13 NMRabsorptions are tabularized in Table I. This preparation is essentiallyhomogeneous by analytical HPLC on Ultrasphere-Octyl® gel with an aqueousammonium acetate and acetonitrile solvent system.

EXAMPLE 5 Separation Of Antibiotic LL-B02964β

A 1182 mg. portion of a mixture of α and β components similar to portion(I) of Example 3 was chromatographed on Bio-Gel® P-4 as described inExample 4. Fractions 55-59, containing antibiotic LL-B02964β accordingto an HPLC analysis conducted as described supra, were combined andfreeze-dried to yield 110 mg. of antibiotic LL-B02964β, having thefollowing characteristics:

Microanalysis (percent): C, 50.91; H, 5.79; N, 13.57; S, 0.00.

Optical Rotation: [α]_(D) ²⁵ =-19±2° (C=0.5%, water).

LL-B02964β produced an infrared absorption spectrum (KBr pellet) asshown in FIG. 6; a U.V. spectrum in water exhibiting absorption maximaat 220 and 256 nm as shown in FIG. 7; ¹ HMR spectra relative totetramethyl silane in ppm, taken as a solution in D₆ dimethyl sulfoxide,as shown in FIG. 8 and under the same conditions with added deuterotrifluoroacetic acid, as shown in FIG. 9; and a ¹³ CNMR spectrumrelative to tetramethyl silane in ppm, taken as a solution in D₆dimethyl sulfoxide, as shown in FIG. 10. The significant ¹³ CNMRabsorptions are tabularized in Table I.

EXAMPLE 6 Analysis Of The Amino Acid Esters Obtained By Acid HydrolysisOf The α, β And γ Antibiotic Components

The individual α, β and γ antibiotics were separately hydrolyzed bydissolving each in 6N hydrochloric acid (1 mg antibiotic to 1 ml acidratio) and heating in a sealed tube for 24 hours at 110° C. The aminoacid residue recovered from the hydrolysis was washed several times withwater while processing on a rotary evaporator which removed excesshydrochloric acid. The residue was then placed under high vacuum for 16hours.

The procedure used to prepare the N-trifluoroacetyl-n-butyl esterderivatives of the amino acid residues was essentially that of Pandey,et. al., J.A.C.S. 99: 8469 (1977). This procedure was carried out oneach of the foregoing antibiotic hydrolysates. A 6 mg. portion of theacid hydrolysate suspended in 1 ml. of 3N hydrochloric acid in 1-butanolwas sealed in a hydrolysis tube and heated at 110° C. for 30 minutes.The reaction mixture was cooled and evaporated to dryness in vacuo. A 2ml. portion of 1-butanoltanol was added to the residue and the mixturewas reevaporated in vacuo. The residue was then heated with 1 ml. ofdichloromethane and 0.5 ml. of trifluorooacetic anhydride at 150° C. for5 minutes in a sealed hydrolysis tube. The mixture was cooled,evaporated under a stream of nitrogen and the residue redissolved in 1ml. of dichloromethane and errevaporated. The residue was taken up in0.3 ml. of dichloromethane for analysis.

The gas chromatographic/mass spectrum analysis was performed under thefollowing conditions: the column used was 3% OV-17 on GAS CHROM®Q(100-200 mesh) (6 feet glass); the detector temperature was 300° C.; theinjector temperature was 250° C.; helium was used as a carrier gas witha flow rate of 30 ml/minute; and the column temperature was programmedto increase from 80° to 300° C. at 15° C./minute and then to hold at300° C. for 10 minutes.

As each ester fraction eluted from the gas chromatograph, an aliquot wasautomatically shunted into a mass spectrumeter which permitteddetermination of the molecular weight of the ester composing thatfraction as well as the fragmentation pattern in the ester. In thismanner, the molecular weights and identities of each of the esters ofthe amino acids present in each antibiotic hydrolysate were determined.Accordingly, the N-trifluoroacetyl-n-butyl esters of amino acidscontained in the polypeptide chain of each antibiotic, LL-BO2964α, β andγ include the following:

β-Alanine Derivative: CF₃ CONHCH₂ CH₂ COOC₄ H₉

Alanine Derivative: CF₃ CONHCH(CH₃)COOC₄ H₉

Glycine Derivative: CF₃ CONHCH₃ COOC₄ H₉

m-Tyrosine Derivative: CF₃ COOC₆ H₄ CH₂ CH(NHCOCF₃)COOC₄ H₉

2-Amino-3-(methylamino) butyric acid Derivative: CH₃N(COCF₃)CH(CH₃)CH(NHCOCF₃)COOC₄ H₉

In addition, three hydantoins formed from (i) alanine and m-tyrosine,(ii) alanine and phenylalanine and (iii) alanine and p-tyrosine werefound in the acid hydrolysates of the α, β and γ componentsrespectively. Uracil was also present in the three acid hydrolysates.

The antibiotic agents of the invention may be administered byconventional routes which would be apparent to those skilled in the art,and in approprate dosage form such as sterile aqueous or alcoholicsolution or suspension, liquid solution, suppository, capsule, tablet,troche, syrup, water, powder, elixir and the like. The agents may begiven alone in the appropriate dosage form or may be combined with asuitable pharmaceutical carrier. Such carriers are well known in the artand include elixirs, excipients, starches, aliphatic alcohols,glycerols, glycols, sugar solids or liquids, polyvinyl pyrolidone,suspension agents, emulsifiers, inert pharmaceutically acceptablediluting powders and liquids, isotonic solutions, gums, gelatins,lubricants, sweeteners, flavoring agents, preservatives, antioxidantsand the like. The methods for preparing such combinations and dosageforms are well known in the art.

In general, the route of administration, dosage form and amount, anddosage rate will be dependent upon the patient's condition and upon thejudgment and observation of his attending physician. An effective amountfor the treatment of bacterial infection or tuberculosis can bedetermined by culturing the infectious organism by known techniques anddetermining the bacteriostatic or bacteriotoxic concentration of agentrequired to inhibit the growth or kill the organism. The physician willthen use these data to determine the requisite parameters ofadministration to the patient. Typically, for antibacterial purposes,from about 30 to about 250 mg. of agent per kg. of patient body weightper day would be administered. For antitubercular treatment, from about30 to 250 mg. of agent per kg. of patent body weight per day would beadministered. Antitubercular treatment will generally require longperiods of administration, typically six months to a year or more andmay also be combined with other methods of therapy, such as use ofisoniazid and the like.

g the bacteriostatic or bacteriotoxic concentration of agent required toinhibit the growth or kill the organism. The physician will then usethese data to determine the requisite parameters of administration tothe patient. Typically, for antibacterial purposes, from about 30 toabout 250 mg. of agent per kg. of patient body weight per day would beadministered. For antitubercular treatment, from about 30 to 250 mg. ofagent per kg. of patent body weight per day would be administered.Antitubercular treatment will generally require long periods ofadministration, typically six months to a year or more and may also becombined with other methods of therapy, such as use of isoniazid and thelike.

We claim:
 1. The antibiotic LL-BO2964α wherein the substantially pureform:(a) exhibits the following significant carbon-13 nuclear magneticresonance absorptions (Abs) in parts per million in dimethylsulfoxide-d₆ relative to the absorption of tetramethyl silane:

    ______________________________________                                        Peak No.                                                                              Abs      Peak No. Abs    Peak No.                                                                             Abs                                   ______________________________________                                        1       13.9     16        96.7  31     137.3                                 2       18.4     17       102.1  32                                           3       27.3     18              33     138.9                                 4                19              34     139.6                                 5       29.3     20       112.9  35     140.4                                 6       30.5     21       113.4  36     150.3                                 7       33.9     22       116.2  37     152.9                                 8       48.5     23              38     156.9                                 9       49.5     24       119.3  39     157.3                                 10      50.4     25       119.9  40     162.7                                 11      55.0     26              41     166.4                                 12      69.9     27       127.6  42     170.1                                 13      71.4     28       128.6  43     171.0                                 14      73.0     29       129.0  44                                           15      91.5     30       135.8  45     173.1                                 ______________________________________                                    

(b) has an optical rotation [α]_(D) ²⁵ of -10° (C being 2.77%, water);(c) has an elemental analysis (percent) of about: C, 50.89; H, 5.67; N,14.44; S, 0.00; (d) has a carbon-13 nuclear magnetic resonance spectrumin dimethyl sulfoxide-d₆ substantially as shown in FIG. 5; (e) producesuracil, alanine, beta-alanine, glycine, m-tyrosine,3-N-methylamino-2-aminobutyric acid and a hydantoin formed from alanineand tyrosine when hydrolyzed by acid; and (f) has an infrared absorptionspectrum in KBr substantially as shown in FIG. 1, an ultravioletspectrum in water substantially as shown in FIG. 2, a proton nuclearmagnetic resonance spectrum in dimethyl sulfoxide-d₆ substantially asshown in FIG. 3, and a proton nuclear magnetic resonance spectrum indimethylsulfoxide-d₆ with added deutero-trifluoroacetic acidsubstantially as shown in FIG.
 4. 2. The antibiotic LL-BO2964β whereinthe substantially pure form:(a) exhibits the following significantcarbon-13 nuclear magnetic resonance absorptions (Abs) in parts permillion in dimethyl sulfoxide-d₆ relative to the absorption oftetramethyl silane:

    ______________________________________                                        Peak No.                                                                              Abs      Peak No. Abs    Peak No.                                                                             Abs                                   ______________________________________                                        1       13.9     16        96.7  31                                           2       18.3     17       102.1  32     138.4                                 3       27.3     18              33     138.9                                 4                19              34                                           5       29.3     20       113.4  35     140.4                                 6       30.4     21              36     150.3                                 7       33.9     22       115.7  37     152.9                                 8       48.9     23              38     156.9                                 9       49.6     24              39     157.3                                 10      50.4     25       119.4  40     162.7                                 11      54.9     26       125.7  41     166.4                                 12      69.4     27       127.7  42     169.9                                 13      71.3     28       129.3  43     171.0                                 14               29              44                                           15      91.4     30              45     173.1                                 ______________________________________                                    

(b) has an optical rotation [α]_(D) ²⁵ of -19±2° (C being 0.5%, water);(c) has an elemental analysis (percent) of about: C, 50.91; H, 5.79; N,13.57; S, 0.00; (d) has a carbon-13 nuclear magnetic resonance spectrumin dimethyl sulfoxide-d₆ substantially as shown in FIG. 10; (e) producesuracil, alanine, beta-alanine, glycine, m-tyrosine,3-N-methylamino-2-aminobutyric acid and a hydantoin formed from alanineand phenylalanine when hydrolyzed by acid; and (f) has an infraredabsorption spectrum in KBr substantially as shown in FIG. 6, anultraviolet spectrum in water substantially as shown in FIG. 7, a protonnuclear magnetic spectrum in dimethyl sulfoxide-d₆ substantially asshown in FIG. 8, and a proton nuclear magnetic resonance spectrum indimethyl sulfoxide-d₆ with added deutero-trifluoroacetic acidsubstantially as shown in FIG.
 9. 3. The antibiotic LL-BO2964γ whereinthe substantially pure form:(a) exhibits the following carbon-13 nuclearmagnetic resonance absorptions (Abs) in parts per million in dimethylsulfoxide-d₆ relative to the absorption of tetramethyl silane:

    ______________________________________                                        Peak No.                                                                              Abs      Peak No. Abs    Peak No.                                                                             Abs                                   ______________________________________                                        1       13.9     16        96.7  31                                           2       18.4     17       102.2  32                                           3       27.4     18       110.3  33     138.9                                 4       28.1     19       111.0  34                                           5       29.3     20       113.4  35     140.4                                 6       30.4     21              36     150.3                                 7       33.9     22       115.7  37     153.0                                 8       48.7     23       118.0  38     156.9                                 9       49.4     24       118.5  39     157.4                                 10      50.4     25       120.5  40     162.7                                 11      54.2     26       123.4  41     166.4                                 12      69.5     27       127.6  42     170.0                                 13      71.4     28       129.0  43     171.1                                 14               29              44     171.9                                 15      91.5     30       135.8  45     173.1                                                                  46     174.9                                 ______________________________________                                    

(b) has an optical rotation [α]_(D) ²⁶ of -20±2° (C being 0.5%, water);(c) has an elemental analysis (percent) of about: C, 52.87; H, 5.75; N,15.10; S, 0.00; (d) has a characteristic carbon-13 nuclear magneticresonance spectrum in dimethyl sulfoxide-d₆ substantially as shown inFIG. 15; (e) produces uracil, alanine, beta-alanine, glycine,m-tyrosine, 3-N-methylamino-2-aminobutyric acid and a hydantoin formedfrom alanine and tyrosine when hydrolyzed by acid; and (f) has aninfrared absorption spectrum in KBr substantially as shown in FIG. 11,an ultraviolet spectrum in water substantially as shown in FIG. 12, aproton nuclear magnetic resonance spectrum in dimethyl sulfoxide-d₆substantially as shown in FIG. 13, and a proton nuclear magneticresonance spectrum in dimethyl sulfoxide-d₆ with addeddeutero-trifluoroacetic acid substantially as shown in FIG.
 14. 4. Apharmacologically acceptable acid addition salt of an antibioticaccording to claim 1, 2, or
 3. 5. A pharmaceutical composition in unitdosage form, useful for treating tuberculosis in warm-blooded animals,comprising a pharmaceutically acceptable carrier in association with aneffective anti-tubercular amount of an antibiotic selected from thegroup consisting of the antibiotic LL-BO2964α as defined in claim 1, theantibiotic LL-BO2964β as defined in claim 2, and the antibioticLL-BO2964γ as defined in claim
 3. 6. A method for treating tuberculosisin warm-blooded animals, comprising administering orally or parenterallyto said animals an effective anti-tubercular amount of an antibioticselected from the group consisting of the antibiotic LL-BO2964α asdefined in claim 1, the antibiotic LL-BO2964β as defined in claim 2, andthe antibiotic LL-BO2964γ as defined in claim
 3. 7. A pharmaceuticalcomposition in unit dosage form, useful for treating bacterialinfections in warm-blooded animals, comprising a pharmaceuticallyacceptable carrier in association with an effective anti-bacterialamount of an antibiotic selected from the group consisting of theantibiotic LL-BO2964α as defined in claim 1, the antibiotic LL-BO2964βas defined in claim 2, and the antibiotic LL-BO2964γ as defined in claim3.
 8. A method of treating bacterial infections in warm-blooded animalscomprising administering orally or parenterally to said animals anantibacterially effective amount of a compound selected from the groupconsisting of the antibiotic LL-BO2964α as defined in claim 1, theantibiotic LL-BO2964β as defined in claim 2, and the antibioticLL-BO2964γ as defined in claim
 3. 9. A process for preparing theantibiotic LL-BO2964α as defined in claim 1, the antibiotic LL-BO2964βas defined in claim 2, or the antibiotic LL-BO2964γ as defined in claim3, which comprises aerobically fermenting a liquid medium at atemperature of about 24°-35° C. for a period of about 40-120 hours, saidmedium containing assimilable sources of carbon, nitrogen and inorganicanions and cations, inoculating said medium with a viable culture ofStreptomyces coeruleorubidus subspecies rubidus NRRL 12372, anantibiotic LL-BO2964α-, LL-BO2964β- or LL-BO2964γ-producing mutantthereof or an ultraviolet-induced mutant strain NRRL 12373, harvesting amash produced thereby and isolating the antibiotics contained in saidmash.